Problems and paradigms

Prokaryotic and eukaryotic chromosomes: what's the difference? Arnold J. Bendich1* and Karl Drlica2 Summary It is widely held that the profound differences in cellular architecture between prokaryotes and eukaryotes, in particular the housing of eukaryotic chromosomes within a nuclear membrane, also extends to the properties of their chromosomes. When chromosomal multiplicity, ploidy, linearity, transcriptional silencing, partitioning, and packaging are considered, no consistent association is found between any of these properties and the presence or absence of a nuclear membrane. Some of the perceived differences can be attributed to cytological limitations imposed by the small size of bacterial nucleoids and the arbitrary choice of representative organisms for comparison. We suggest that the criterion of nucleosome-based packaging of chromosomal DNA may be more useful than the prokaryote/eukaryote dichotomy for inferring the broadest phylogenetic relationships among organisms. BioEssays 22:481±486, 2000. ß 2000 John Wiley & Sons, Inc. Introduction It has long been customary to divide organisms into two groups, the prokaryotes, whose DNA intimately interacts with cytoplasm, and the eukaryotes, whose DNA is separated from cytoplasm by a nuclear membrane. Eukaryotic chromosomes generally change from a diffuse form to a highly condensed one during mitosis, and chromosomal DNA, which has specialized ends (telomeres), is usually packaged into nucleosomes by histones. Ploidy levels can differ widely, and chromosomal regions, or even whole chromosomes, can be silenced by compacting parts of the genome through heterochromatinization. In contrast, prokaryotic chromosomes have often been thought to lack those properties; thus, it has been convenient to relate the differences to the presence or absence of a nuclear membrane. Recent studies, however, reveal that prokaryotic chromosomes, or at least portions of them, undergo large movements similar to those found in eukaryotic mitosis (reviewed in Refs. 1,2); moreover, examples of histone-based DNA packaging have

1

Departments of Botany and Genetics, University of Washington, Seattle, Washington. 2 Public Health Research Institute, 455 First Avenue, New York, New York.  Correspondence to: Arnold J. Bendich, Department of Botany, University of Washington, Seattle, WA 98195-5325.

BioEssays 22:481±486, ß 2000 John Wiley & Sons, Inc.

been found in some prokaryotes (reviewed in Refs. 3,4). By blurring the distinction between eukaryotic and prokaryotic chromosomes, these observations raise the possibility that the two groups might not differ categorically in terms of other chromosomal features. To examine distinctions between prokaryotic and eukaryotic chromosomes we have surveyed a large number of species. We found so many exceptions to commonly held views about chromosome multiplicity, ploidy, linearity, heterochromatinization, partitioning, and histone-based DNA packaging that we were forced to conclude that chromosomal properties do not correlate well with the presence or absence of a nuclear membrane. The fading prokaryotic/eukaryotic dichotomy means that some other character(s) should be used if we are to divide organisms into two groups. DNA packaging strategies could serve as such a character. Identifying homologous features of chromosome structure and function among organisms would then take on new meaning. Below, we begin an argument for a nucleosomal/ non-nucleosomal dichotomy by pointing out counter-examples to traditional prokaryotic/eukaryotic distinctions (summarized in Table 1). Chromosome number and genome size Prokaryotes are frequently thought to contain only a single chromosome. Small size, however, has usually precluded a direct count of chromosomes in prokaryotic cells. Recently, chromosome number has been inferred from DNA fragment mapping and pulsed-field gel electrophoresis, techniques that have generated a growing list of species with two or more chromosomes (in addition to very large plasmids). The number of large, circular-mapping chromosomes is 2 for Vibrio species,(5,6) Deinococcus radiodurans,(7) Rhodobacter sphaeroides, Leptospira interrogans, and Brucella species, three for Rhizobium meliloti (Ref. 8 and references therein), and 2-4 among isolates of Burkholderia (Pseudomonas) cepacia.(9) Some Agrobacterium species contain one circular- and one linear-mapping chromosome,(8) and Paracoccus denitrificans contains three unmapped chromosomes.(10) The chromosome number in prokaryotes can even exceed that in eukaryotes, since the number is only one (2n ˆ 2) in the ant Myrmecia pilosula.(11) Prokaryotes can also have a larger genome size. For example, the genomes of the prokaryotes Myxococcus

BioEssays 22.5

481

Problems and paradigms

Table 1. Counter-Examples of Properties Traditionally Associated with the Presence of a Nuclear Membrane Multiple chromosomes Bacteria with two or more chromosomes: Vibrio species Brucella species Deinococcus radiodurans Rhizobium meliloti Rhodobacter sphaeroides Burkholderia cepacia Leptospira interrogans Paracoccus denitrificans Eukaryotes with only one chromosome: Myrmecia pilosula Large genomes Bacterial genomes (Myxococcus xanthus; Calothrix strains) can be large than those of some eukaryotes (Encephalitozoon species; Pneumocystis carinii) Polyploidy in bacteria: Escherichia coli Methanococcus jannaschii Deinococcus radiodurans Desulfovibrio gigas

Polyploidy Azotobacter vinlandii Borrelia hermsii Synechococcus PCC 6301 Epulopiscium fishelsoni

Linear chromosomes Bacteria with linear chromosomes: Agrobacterium tumefaciens C58 Borrelia burgdorferi a Rhodococcus fasciens Streptomyces speciesa Eukaryotes with circular chromosomes: Schizosaccharomyces pombe (telomerase-deficient cells) Transcriptional silencing, heterochromatinization Prokaryotic silencing: E. coli plasmids (regions of P1 and F) Bacillus species (spore) Chlamydia trachomatis (extracellular elementary body) Halobacterium salinarium (late exponential growth) Caulobacter crescentus (swarmer cell) Epulopiscium fishelsoni (condensed nucleoid) Forms of Eukaryotic Mitosis Undetectable metaphase plate: Saprolegnia ferax Nanochlorum eucaryotum Aggregata eberthi Aulacantha scolymantha Undetectable chromosomal fibers: N. eucaryotum hypermastigidsb A. scloymanthab dinoflagellatesb,c tricomonadsb Undetectable spindle pole structures: A. scolymantha higher plants Trypanosoma many Protozoa Chromosomes permanently condensed: dinoflagellatesd euglenoids hypermastigidsc Chromosomes permanently decondensed: Zygorhynchus moelleri N. eucaryotum Binary nuclear division: N. eucaryotum A. scolymantha Histones and Nucleosomes Eukaryotes with no histones or nucleosomes: many dinoflagellates N. eucaryotum Prokaryotes with histones and nucleosomes: Methanothermus fervidus Methanobacterium thermoautotrophicum a

Telomeric ends on DNA. Spindle fibers are present but may not directly move chromosomes. Some species. d Most species. b c

482

BioEssays 22.5

xanthus (9.5 Mb of DNA(12)) and Calothrix strains (12 Mb;(13)) are larger than those of the eukaryotes Encephalitozoon species (2.3 and 2.9 Mb(14)) and Pneumocystis carinii (8.8 Mb(15)). Assuming a density of one open reading frame per 1.1 kb of prokaryotic chromosomal DNA, the number of different genes in M. xanthus and Calothrix exceeds the 5900 in baker's yeast.(16) It is evident that neither chromosome number nor genome size (nor probably gene number) strictly correlates with the presence or absence of a nuclear membrane. Chromosome copy number Metazoans and plants are typically diploid, although ploidy can exceed 500 in certain tissues such as salivary glands in some insects and endosperm in maize.(17) In contrast, prokaryotes are thought to be genetically monoploid. The copy number of genomic DNA can be higher in prokaryotes, however, than in most animal and plant tissues. For example, the number of genome equivalents per Escherichia coli cell is about 11 (the range is about 5±18) during early exponential growth in a rich medium and 2, 4, or 8 during stationary phase.(18) The average chromosomal copy number for Methanococcus jannaschii is about seven (the range is about 3±15) during exponential growth and about three (range 1±5) in stationary phase.(19) D. radiodurans is similar: the chromosomal copy number is 10 during exponential growth and four during stationary phase.(20) Copy numbers can reach about eight (the range is 3±18) for the cyanobacterium Synechococcus PCC 6301,(21) 9-17 for Desulfovibrio gigas(22) and 16 for Borrelia hermsii.(23) For Azotobacter vinlandii , the copy number can increase from 4 to 40 to >100 as the culture progresses from early exponential through late exponential to stationary phase.(24) The most dramatic case among either prokaryotes or eukaryotes occurs in the eubacterium Epulopiscium fishelsoni: the DNA content varies by 4 to 5 orders of magnitude among individuals at different stages of the life cycle, and it can greatly exceed the amount of DNA found in mammalian nuclei.(25) These examples show that high genome copy number and variable ploidy among individual cells are not restricted to eukaryotes. Indeed, these properties might even be more common in prokaryotes but the monoploid nature of prokaryotic genetics in model organisms has hidden them from us. Circular or linear form and telomeres The famous autoradiographic image presented by Cairns,(26) which shows a circular replicating E. coli chromosome, appeared to confirm earlier genetic mapping data in this species and provided the foundation for the belief that prokaryotic chromosomes, being circles, are fundamentally different from the linear structures found in eukaryotes. Linear chromosomes, however, can occur in bacteria. Species with linear chromosomes include Borrelia burgdor-

Problems and paradigms feri, Rhodococcus fasciens, Agrobacterium tumefaciens C58, and Streptomyces species (Ref. 8 and references therein). Moreover, the ends of these bacterial chromosomes resemble eukaryotic chromosomal telomeres, structures that are designed to solve the ``ends problem'' of linear DNA replication (Ref. 27 and references therein). For example, chromosomal ends in B. burgdorferi are hairpin structures. In Streptomyces, the ends contain several short, inverted, repetitive sequences capable of forming hairpins, and attached to the 5 0 termini are proteins that probably prime synthesis complementary to the 3 0 ends of the DNA. Thus, not all bacteria have circular chromosomes. Since so few species have been analyzed adequately, it is not even clear that the circle is the most common chromosomal form among prokaryotes. Furthermore, the evidence for circularity for most bacterial chromosomes is based exclusively on mapping data, which alone cannot distinguish between circular and linear chromosomes (for example, the circular map of bacteriophage T4 DNA arises from circular permutation of a linear DNA). Even the circular form found by Cairns may not be representative, since nearly all of the molecules revealed by his technique were uninterpretable tangles and linear forms.(28) In eukaryotic cells, chromosomes are thought to be linear. However, exclusively circular forms can be maintained under certain conditions: the most common survivors among telomerase-deficient cells of fission yeast circularized their chromosomes.(29) Thus the circular/linear form of chromosomes does not necessarily correlate with the prokaryotic/ eukaryotic form of cellular architecture. Transcriptional silencing and heterochromatin Not all genetic information carried by an organism is used continuously throughout its life cycle, and to ensure inactivation, some genomic regions are transcriptionally silenced by compaction with proteins. Eukaryotic silencing refers to the blocking of gene expression that spreads from a regulatory site on DNA, the silencer.(30) Silencing may be synonymous with formation of highly compacted heterochromatin.(30) Silencing also occurs in E. coli, spreading over a distance of about 10 kb from the parS site on plasmid P1 (31) and the sopC site on the F plasmid.(32) As in eukaryotes, silencing in bacteria requires particular proteins (ParB and SopB for these cases in E. coli). A compacted DNA-protein complex can be recognized cytologically if it is large enough to bind sufficient Giemsa, Feulgen or other cytological stain. Such is the case in eukaryotes having large nuclear genomes in which silenced DNA can be recognized as heterochromatin, either as parts of chromosomes or as whole, compacted chromosomes (``B'' chromosomes, for example; see Ref. 17). In some organisms, however, such as baker's yeast and prokaryotes, the

genome is too small or there is too little silenced DNA for detection by staining in situ. Nevertheless, a general shutdown of genomic activity that is correlated with chromosomal DNA compaction does occur in bacteria. For example, during sporulation in Bacillus the chromosome is bound with new proteins as its transcriptional activity ceases.(33) Similarly, a histone H1-like protein in Chlamydia trachomatis probably causes chromosomal condensation during the conversion of the metabolically-active reticulate body to the inactive, extracellular elementary body form (Ref. 34 and references therein). A third case is observed when the cells of the archaebacterium Halobacterium salinarium progress from early to late exponential phase of growth. The nucleoid changes from a form containing a naked DNA to one having a beads-on-string appearance typical of nucleosomal DNA; this change is also reflected in nucleoid sedimentation properties.(35) In another example, formation of swarmer cells of Caulobacter crescentus is associated with cessation of DNA replication, and the nucleoid appears to be in a compacted form when analyzed by sedimentation and electron microscopy.(36) After differentiation from the swarmer to the stalked cell form, DNA replication begins, and the nucleoid changes to a more open structure, possibly reflecting an activation of the genome. Finally, fluorescence measurements of DNA and RNA within enormous prokaryotic cells (up to 500 microns long) of E. fishelsoni suggest that decondensation and dispersion of the nucleoid is accompanied by increased transcriptional activity.(25) In their condensed state, these nucleoids resemble condensed eukaryotic chromosomes of dinoflagellates. Thus both prokaryotes and eukaryotes employ protein-mediated compaction strategies to activate and inactivate DNA. The apparent differences may be due to limitations of cytological analysis. It will now be interesting to look for homologies among the proteins involved. Such homologies, if found, could support the notion that chromosome structure represents a deeper evolutionary dichotomy than the presence or absence of nuclear membranes. Forms of mitosis The standard mitosis so familiar to most of us includes condensation and alignment of chromosomes at a metaphase plate, the movement of chromosomes to cell poles along spindle fibers emanating from centrioles or other structures at the spindle poles, and finally chromosomal decondensation after cytokinesis. Mitosis is enormously diverse among eukaryotes, however. For example, in Saprolegnia ferax, an oomycete fungus, and Aggregata eberthi, a member of the Sporozan class of Protozoa, mitosis is accomplished without the chromosomes appearing at a typical metaphase plate.(37,38) For Aulacantha scolymantha, a member of the Rhizopoda, the chromosomes also move to the poles without prior arrangement at a metaphase plate,

BioEssays 22.5

483

Problems and paradigms and they do so without recognizable spindle fiber attachments to the chromosomes or spindle pole structures.(38,39) Indeed, the absence of structure at the spindle poles is a feature of many Protozoa, including the flagellate Trypanosoma.(38) It is also a feature of higher plants.(40) In trichomonads, hypermastigids, and some dinoflagellates, microtubules participate in nuclear divsion, but they do not appear to be connected directly to the chromosomes.(38,39) Instead, microtubules are attached to the outside of the nuclear membrane at the points where the chromosomal kinetochores are permanently attached to the inside of that membrane.(38,39) The chromosomes of some eukaryotes also fail to undergo condensation-decondensation during the cell cycle. For example, chromosomes are permanently condensed in most dinoflagellates, euglenoids, and some hypermastigids.(41,42) Conversely, chromosomes are diffuse throughout nuclear division in the fungus Zygorhynchus moelleri (43) and the green alga Nanochlorum eucaryotum.(44) In the case of N. eucaryotum, the nucleus divides by simply pinching in two. No spindle fibers are seen. Indeed, the binary division of the nucleus in N. eucaryotum and in A. scolymantha more closely resembles the binary division of the enormous prokaryotic nucleoid of E. fishelsoni than the mitosis found in higher animals and plants. In view of such mechanistic diversity, the choice of a ``standard'' form of eukaryotic mitosis is clearly arbitrary, making it unclear which system to choose in order to contrast eukaryotes with prokaryotes. Until recently, lack of clear choice did not matter, since a process comparable to mitosis was unknown in bacteria. We now realize that the failure to detect mitotic-like events has been due to cytological limitations imposed by small cell size: forms of mitosis have been demonstrated in B. subtilis, E. coli, and C. crescentus using fluorescence microscopy.(1,2,45) The key observation is the rapid, cell cycledependent movement of the replication origin (oriC) region of chromosomes to opposite poles of the cell. Moreover, a region near the terminus of replication moves from a polar to a midcell position. To facilitate oriC movement, the bacterial chromosome appears to have both partitioning regions near oriC and proteins that bind to these regions. In B. subtilis, ten related, 8 bp inverted repeat DNA sequences are scattered across about 800 kb of the 4200 kb chromosome; eight of these are bound in vivo to Spo0J, a protein involved in partitioning chromosomes to daughter cells.(46) Such a distribution of bacterial centromere-like DNA elements is similar to the most common type of distribution of functional centromeric DNA in eukaryotes, the CEN-containing regional centromere.(47) Although additional work is needed to determine whether prokaryotes generally have a regional centromere or a point centromere (about 200 bp, found thus far among eukaryotes only in budding yeasts(47)), it is apparent that at least some prokaryotic centromeres

484

BioEssays 22.5

structurally resemble eukaryotic centromeres. Indeed, it is difficult to explain chromosomal segregation in bacterial species that have more than one chromosome without invoking a mitotic-like process.(1) Microtubule-containing fibers have not been found attached to bacterial chromosomes, thus distinguishing chromosome partitioning in prokaryotes from that in most eukaryotes. It is possible, however, that the movement of bacterial chromosomes instead requires attachment to a movable membrane,(48) as proposed for chromosomal propulsion in closed extranuclear pleuromitosis.(39) The bacterial process might then be homologous to one of the six types of eukaryotic mitosis.(39) Many more experiments are required to develop this idea, since the possibility still exists that the force moving bacterial chromosomes is provided by the activity of DNA polymerase(45) rather than by a post-replicative process as observed for eukaryotes. It is also possible that many types of bacterial ``mitosis'' exist, with the consequence that homology searches among the mitosis proteins may be difficult. Histones, nucleosomes, and chromosomal compaction Chromosomal compaction in most eukaryotes involves the wrapping of DNA around histones to form nucleosomes. This is not true for all eukaryotes, however. Despite their permanently condensed chromosomes, many dinoflagellates contain neither histones nor nucleosomes.(42) Similarly, neither histones nor nucleosomes are detectable in the green alga N. eucaryotum.(44) With respect to prokaryotes, some (but not all) archaebacteria contain histones that wrap DNA into structures that visibly resemble nucleosomes.(3,49) Other prokaryotes, such as E. coli, have neither histones nor nucleosomes, although they do contain basic proteins such as HU and IHF that bend DNA sharply (wrapping of DNA within eubacteria is still being investigated). The smooth, extended non-nucleosomal appearance of DNA fibers from these bacteria shows that the forces that compact DNA in vivo are not maintained after extraction. The 1100 micronlong chromosome from E. coli in Cairns'(26) image also illustrates this point, since it was prepared in the absence of detergent or protease. In summary, examples exist for both prokaryotes and eukaryotes in which histones compact DNA into nucleosomes and examples in which both histones and nucleosomes are absent. For non-nucleosomal organisms, the central question concerns how their DNA is compacted. An attractive idea is that compacting forces arise through macromolecular crowding due to a high cytoplasmic concentration of non-DNAbinding proteins and other large molecules, as shown in vitro with cytoplasmic extracts of E. coli (Ref. 50 and references therein). The chromosomes in some organisms may be packaged by both nucleosomes and crowding, perhaps at

Problems and paradigms the same time or at different times in the life cycle. Although we have speculated about the consequences of the crowding type of DNA packaging,(51) crowding has not yet been demonstrated in vivo.(52)

organisms or convergent evolution. In either case, it has become apparent that the principal character currently used to distinguish bacterial cells from higher life forms, the nuclear membrane, is not a very good discriminator.

Conclusion It is widely held that most eukaryotes differ from most eubacteria with respect to the chromosomal properties considered above. This is undoubtedly true for some of these properties. The counter-examples discussed above, however, show that there is no obligatory association between any of these chromosomal properties and the presence or absence of a nuclear membrane. Some of the apparent prokaryotic/eukaryotic chromosomal differences can be attributed to difficulties in analyzing structural features in situ with tiny bacterial nucleoids and to the arbitrary choice of representative organisms for comparison. Consequently, prokaryotic and eukaryotic organisms may not be as different as is generally believed. A dichotomy among organisms can be seen, however, when nucleosome-based packaging is considered, provided that one accepts the conclusion that the absence of nucleosomes in some dinoflagellates(53) and N. eucaryotum (54) represents loss of that character. In this dichotomy, some members of the Euryarchaeota are grouped with the eukaryotes and are distinguished from non-nucleosomal cell types, which include most prokaryotes that have been examined. The distinction also applies to the presence, in nucleosomal organisms, of histones because non-nucleosomal organisms lack these proteins.(3,4) The presence of histones and nucleosomal packaging of chromosomal DNA probably predates the development of a nuclear membrane, and so distinguishing chromosomes on the basis of DNA packaging is likely to reflect a deeper phylogenetic branching. This view focuses attention on delineating nucleosomal archaea from non-nucleosomal groups. Such characterization, which is just beginning, may be important for identifying the modern representatives of the archaea likely to have participated in the origin of eukaryotes. For example, Gupta(55) proposed that eukaryotes originated from a fusion between a member of the Crenarchaeota and a Gram-negative-like eubacterium; however, the archaea in which nucleosomes have been identified are in a different group, the Euryarchaeota. In contrast, eukaryotic origin models involving symbiosis do specify the Euryarchaeota as one of the symbionts.(56) Thus, chromosome structure may be a useful character for phylogenetic inference. Finally, we note that bacterial species in the order Planctomycetales and bacteria-like sponge symbionts (some having an archaea-like cell wall appearance) contain a nucleoid bounded by a single(57±59) or a double(60) bilayer membrane. It is now important to determine whether such membranes represent homologies between lower and higher

References

1. Margolin W. A green light for the bacterial cytoskeleton. Trends Microbiol 1998;6:233±238. 2. Sharpe ME, Errington J. Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism. Trends Genet 1999;15:70±74. 3. Sandman K, Pereira SL, Reeve JN. Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome. Cell Mol Life Sci 1998;54: 1350±1364. 4. Li JY, Arnold-Schulz-Gahmen B, Kellenberger E. Histones and histonelike DNA-binding proteins: correlations between structural differences, properties and functions. Microbiology 1999;145:1±2. 5. Trucksis M, Michalski J, Deng YK, Kaper JB. The Vibrio cholerae genome contains two unique circular chromosomes. Proc Natl Acad Sci USA 1998;95:14464±14469. 6. Yamaichi Y, Iida T, Park KS, Yamamoto K, Honda T. Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species. Mol Microbiol 1999;31:1513±1521. 7. Lin J, Qi R, Aston C, Jing J, Anantharaman TS, Mishra B, White O, Daly MJ, Minton KW, Venter JC. Whole-genome shotgun optical mapping of Deinococcus radiodurans. Science 1999;285:1558±1562. 8. Jumas-Bilak E, Michaux-Charachon S, Bourg G, Ramuz M, AllardetServent A. Unconventional genomic organization in the alpha subgroup of the Proteobacteria. J Bacteriol 1998;180:2749±55. 9. Lessie TG, Hendrickson W, Manning BD, Devereux R. Genomic complexity and plasticity of Burkholderia cepacia. FEMS Microbiol Lett 1996;144:117±128. 10. Winterstein C, Ludwig B. Genes coding for respiratory complexes map on all three chromosomes of the Paracoccus denitrificans genome. Arch Microbiol 1998;169:275±281. 11. Crosland MWJ, Crozier RH. Myrmecia pilosula, an ant with only one pair of chromosomes. Science 1986;231:1278±1281. 12. Chen HW, Kuspa A, Keseler IM, Shimkets LJ. Physical map of the Myxococcus xanthus chromosome. J Bacteriol 1991;173:2109±2115. 13. Herdman M, Janvier M, Rippka R, Stanier RY. Genome size of Cyanobacteria. J Gen Microbiol 1979;111:73±85. 14. Peyretaillade E, Biderre C, Peyret P, Duffieux F, Metenier G, Gouy M, Michot B, Vivares CP. Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core. Nucleic Acids Res 1998;26:3513±3520. 15. Wyder MA, Rasch EM, Kaneshiro ES. Quantitation of absolute Pneumocystis carinii nuclear DNA content. Trophic and cystic forms isolated from infected rat lungs are haploid organisms. J Eukaryot Microbiol 1998;45:233±239. 16. Doolittle RF. Microbial genomes opened up. Nature 1998;392:339±342. 17. John. B, Miklos GLG. The eukaryote genome in development and evolution. London: Allen and Unwin; 1988. p 416 (215 and 288). 18. Akerlund T, Nordstrom K, Bernander R. Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli. J Bacteriol 1995;177:6791±6797. 19. Malandrin L, Huber H, Bernander R. Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome. Genetics 1999;152:1315±1323. 20. Hansen MT. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J Bacteriol 1978;134:71±75 21. Binder BJ, Chisholm SW. Relationship between DNA cycle and growth rate in Synechococcus sp. strain PCC 6301. J Bacteriol 1990;172: 2313±2319. 22. Postgate JR, Kent HM, Robson RL, Chesshyre JA. The genomes of Desulfovibrio gigas and D. vulgaris. J Gen Microbiol 1984;130: 1597±1601. 23. Kitten T, Barbour AG. The relapsing fever agent Borrelia hermsii has multiple copies of its chromosome and linear plasmids. Genetics 1992; 132:311±324.

BioEssays 22.5

485

Problems and paradigms

24. Maldonado R, Jimenez J, Casadesus J. Changes of ploidy during the Azotobacter vinelandii growth cycle. J Bacteriol 1994;176: 3911±3919. 25. Bresler V, Montgomery WL, Fishelson L, Pollak PE. Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA. J Bacteriol 1998;180: 5601±5611. 26. Cairns J. The chromosome of Escherichia coli. Cold Spring Harbor Symp Quant Biol 1963;28:43±46. 27. Casjens S, Murphy M, DeLange M, Sampson L, van Vugt R, Huang WM. Telomeres of the linear chromosomes of Lyme disease spirochaetes: nucleotide sequence and possible exchange with linear plasmid telomeres. Mol Microbiol 1997;26:581±596. 28. Cairns J. A minimun estimate for the length of the DNA of Escherichia coli obtained by autoradiography. J Mol Biol 1962;4:407±409. 29. Nakamura TM, Cooper JP, Cech TR. Two modes of survival of fission yeast without telomerase. Science 1998;282:493±496. 30. Rine J. On the mechanism of silencing in Escherichia coli. Proc Natl Acad Sci USA 1999;96:8309±8311. 31. Rodionov O, Lobocka M, Yarmolinsky M. Silencing of genes flanking the P1 plasmid centromere. Science 1999;283:546±549. 32. Kim SK, Wang JC. Gene silencing via protein-mediated subcellular localization of DNA. Proc Natl Acad Sci USA 1999;96:8557±8561. 33. Setlow P. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu Rev Microbiol 1995;49:29±54. 34. Christiansen G, Pedersen LB, Koehler JE, Lundemose AG, Birkelund S. Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA. J Bacteriol 1993;175:1785±1795. 35. Takayanagi S, Morimura S, Kusaoke H, Yokoyama Y, Kano K, Shioda M. Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium. J Bacteriol 1992;174:7207±7216. 36. Brun YV, Marczynski G, Shapiro L. The expression of asymmetry during Caulobacter cell differentiation. Annu Rev Biochem 1994;63: 419±450. 37. Heath IB. Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax. J Cell Biol 1980;84:531±546. 38. Grell KG. Protozoology. New York: Springer-Verlag; 1973. 554 p. 39. Raikov IB. The protozoan nucleus, morphology, and evolution. Cell Biology Monographs, Wien, New York: Springer-Verlag; 1982. 474 p. 40. Vaughn KC, Harper JD. Microtubule-organizing centers and nucleating sites in land plants. Int Rev Cytol 1998;181:75±149. 41. Sleigh MA. Protozoa and other protists. Cambridge: Cambridge University Press; 1989. 342 p. 42. Rizzo PJ. The enigma of the dinoflagellate chromosome. J Protozool 1991;38:246±252. 43. Heath IB, Rethoret K. Mitosis in the fungus Zygorhynchus moelleri: evidence for stage specific enhancement of microtubule preservation by freeze substitution. Eur J Cell Biol 1982;28:180±189.

486

BioEssays 22.5

44. Zahn RK. A green alga with minimal eukaryotic features: Nanochlorum eucaryotum. Origins of Life 1984;13:289±303. 45. Gordon GS, Wright A. DNA segregation: putting chromosomes in their place. Curr Biol 1998;8:R925±R927. 46. Lin DC, Grossman AD. Identification and characterization of a bacterial chromosome partitioning site. Cell 1998;92:675±685. 47. Clarke L. Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes. Curr Opin Genet Dev 1998;8:212±218. 48. Van Helvoort JM, Huls PG, Vischer NO, Woldringh CL. Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB(Ts) filaments after release from chloramphenicol inhibition. Microbiology 1998;144:1309±1317. 49. Periera SL, Grayling RA, Lurz R, Reeve JN. Archeal nucleosomes. Proc Natl Acad Sci USA 1997;94:12633±12637. 50. Zimmerman SB, Murphy LD. Macromolecular crowding and the mandatory condensation of DNA in bacteria. FEBS Lett 1996;390:245±248. 51. Drlica K, Bendich AJ. Chromosome, Bacterial, In: Encylcopedia of Microbiology, J Lederberg, editor. San Diego: Academic Press; 2000. p C±162±C±175. 52. Hildebrandt ER, Cozzarelli NR. Comparison of recombination in vitro and in E. coli cells: measure of the effective concentration of DNA in vivo. Cell 1995;81:331±340. 53. Sala-Rovira M, Geraud ML, Caput D, Jacques F, Soyer-Gobillard MO, Vernet G, Herzog M. Molecular cloning and immunolocalization of two variants of the major basic nuclear protein (HCc) from the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta). Chromosoma 1991; 100:510±518. 54. Schreiner M, Geisert M, Oed M, Arendes J, Gungerich U, Breter HJ, Stuber K, Weinblum D. Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences. J Mol Evol 1995;40:428±442. 55. Gupta RS. Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev 1998;62:1435±1491. 56. Lopez-Garcia P, Moreira D. Metabolic symbiosis at the origin of eukaryotes. Trends Biochem Sci 1999;24:88±93. 57. Fuerst JA, Webb RI, Garson MJ, Hardy L, Reiswig HM. Membranebounded nucleoids in microbial symbionts of marine sponges. FEMS Microbiol Lett 1998;166:29±34. 58. Fuerst JA, Webb RI, Garson MJ, Hardy L, Reiswig HM. Membranebounded nuclear bodies in a diverse range of microbial symbionts of great barrier reef sponges. Memoirs Queensland Museum 1999;44: 193±203. 59. Strous M, Fuerst JA, Kramer EH, Logemann S, Muyzer G, van de PasSchoonen KT, Webb R, Kuenen JG, Jetten MS. Missing lithotroph identified as new planctomycete. Nature 1999;400:446±449. 60. Fuerst JA, Webb RI. Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus. Proc Natl Acad Sci USA 1991;88:8184±8188.